Injectable, Non-Aqueous Suspension with High Concentration of Therapeutic Agent

ABSTRACT

An injectable, nonaqueous suspension including at least one therapeutic agent suspended in a single component vehicle. The single component vehicle is a single amphiphilic material, such as a polyethoxylated castor oil or derivative thereof a polyoxyethylene alkyl ether, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene stearate, a block copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, a block copolymer of polypropylene oxide-polyethylene oxide-polypropylene oxide, a tetra-functional block copolymer of polyethylene oxide-polypropylene oxide, or a tetra-functional block copolymer of polypropylene oxide-polyethylene oxide. A dosage kit that includes the injectable, nonaqueous suspension and a method of administering the injectable, nonaqueous suspension are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a utility conversion and claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Patent Application No. 60/897,643filed Jan. 26, 2007, entitled “INJECTABLE, NONAQUEOUS SUSPENSION WITHHIGH CONCENTRATION OF THERAPEUTIC AGENT.”

TECHNICAL FIELD

The present invention relates to an injectable suspension that includesa therapeutic agent. More specifically, the present invention relates toan injectable, nonaqueous suspension that includes the therapeutic agentsuspended in a single component vehicle.

BACKGROUND

Proteins and peptides have become powerful therapeutic agents in thetreatment of various diseases, such as cancer, inflammatory,cardiovascular, respiratory, and infectious diseases. However,formulation and delivery of these molecules are challenging due tosolubility and viscosity limitations. Except for highly potentmolecules, formulations of these molecules need to contain relativelyhigh concentrations of the protein to enable efficacious dose levels bysubcutaneous (“SC”) or intramuscular (“IM”) routes of administration.

Commercialization strategies often involve lyophilized formulations thatrequire reconstitution of the protein prior to being delivered byinjection, which can add costs and time to the manufacturing process.Ready-to-use solution formulations of proteins and peptides, whenfeasible, can minimize this inconvenience. However, the requirement fora high concentration of the protein adds complexity to formulationdesign and promotes instability.

It has been estimated that greater than 20% of all biopharmaceuticalscurrently being evaluated in clinical trials are monoclonal antibodies(“mAbs”). In general, mAb therapies require the delivery of betweenapproximately 100 mg and approximately 1 g of protein per dose. Becausethe high end of formulation concentrations for mAbs is typically in therange of 50 mg/ml, such treatments commonly require the administrationof 2 to 20 ml. Typically, such volumes are administered only throughintravenous (“IV”) infusion performed in a clinical or hospital setting,which leads to poor patient compliance.

To achieve a high protein concentration, nonaqueous suspensions ofproteins have been formulated. In these formulations, the protein issuspended in a vehicle that includes at least two of the following: apolymer, a surfactant, and a solvent. The formulation has a proteinconcentration of up to 500 mg/ml.

To expand therapeutic opportunities and increase patient compliance, amethod of achieving a high concentration of mAbs is needed so that largeprotein doses are deliverable in a small volume appropriate for SC or IMinjection. One possible approach is to prepare extremely highconcentration preparations of soluble mAbs, on the order of 150 to 250mg/ml. However, achieving such highly concentrated mAb solutions isproblematic due to solubility limitations and/or relatively highviscosities, which often results in protein aggregation and poor overallstability.

Polyethoxylated castor oil (also known as polyoxyl castor oil, polyoxyl35 castor oil, polyoxyethylated castor oil, macogolglycerol ricinoleate,or macrogolglyceroli ricinoleas) has been used as a solvent forpharmaceutical compositions that include a hydrophobic drug, such asmiconazole, echinomycin, teniposide, diazepam, althesin, or paclitaxel.The hydrophobic drug and the polyethoxylated castor oil form a solution.To further solubilize the hydrophobic drug, many of these pharmaceuticalcompositions also include an alcohol.

There remains a need to develop highly concentrated protein formulationsto enable delivery of a variety of therapeutic proteins in a convenientway with a small volume.

SUMMARY OF THE INVENTION

The present invention relates to an injectable, nonaqueous suspensionthat includes at least one therapeutic agent suspended in a singlecomponent vehicle.

The present invention also relates to a dosage kit that includes asyringe and the injectable, nonaqueous suspension.

The present invention also relates to a method of administering atherapeutic agent that includes suspending the at least one therapeuticagent in the single component vehicle and injecting the injectable,nonaqueous suspension into a patient in need thereof.

BRIEF DESCRIPTION OF DRAWINGS

While the specification concludes with claims particularly pointing outand distinctly claiming that which is regarded as the present invention,the advantages of this invention may be more readily ascertained fromthe following description of the invention when read in conjunction withthe accompanying drawings in which:

FIG. 1 is a graph showing in vitro release data for a bovine serumalbumin (“BSA”)/Cremophor® ELP formulation;

FIG. 2 is a graph showing injection force data for a BSA/Cremophor® ELPformulation;

FIG. 3 is a graph showing injection force data for a BSA/Cremophor® RH40 formulation;

FIG. 4 is a graph showing stability data for a monoclonal antibody(“mAb”) CNTO 1275/Cremophor® ELP formulation; and

FIG. 5 is a graph showing biocompatibility data for Cremophor® ELP.

DETAILED DESCRIPTION OF THE INVENTION

An injectable, nonaqueous suspension having at least one therapeuticagent and a single component vehicle is disclosed. The therapeutic agentis suspended in the single component vehicle. The injectable, nonaqueoussuspension may be formulated to administer a high dose of thetherapeutic agent in a small dose volume. For instance, the dose volumemay be less than or equal to approximately 2 ml/injection. Theinjectable, nonaqueous suspension may provide immediate delivery of alow potency, therapeutic agent.

As used herein, the term “therapeutic agent” refers to a compound thatprovides a desired biological or pharmacological effect whenadministered to a human or animal. The therapeutic agent may be presentas a solid in a dosage form of the injectable, nonaqueous suspension.The therapeutic agent may be minimally soluble or swellable in thesingle component vehicle, maintaining the therapeutic agent in asubstantially solid form in the injectable, nonaqueous suspension. Thesolubility of the therapeutic agent in the injectable, nonaqueoussuspension may be less than approximately 1% by weight (“wt %”), such asless than approximately 0.5 wt % or less than approximately 0.1 wt %.The therapeutic agent remains suspended in the injectable, nonaqueoussuspension independent of the therapeutic agent's molecular structure ormolecular weight. For the sake of example only, the therapeutic agentmay be a small molecule, protein, antibody, mimetibody, mAb, antibodyfragment (including a diabody, triabody, or tetrabody), peptide, enzyme,nucleotide, DNA fragment, RNA fragment, plasmid fragment, nucleotidefragment, or mixtures thereof. In one embodiment, the therapeutic agentis a mAb, such as CNTO 1275 or CNTO 148. CNTO 1275 is a human mAb toanti-IL-12/23p40, and is described in U.S. Pat. No. 6,902,734, thecontents of which are hereby incorporated herein by this reference. CNTO148 is an antibody against human TNF-a, and is described in U.S. patentapplication Ser. No. 09/920,137, filed Aug. 1, 2001, entitled “Anti-TNFAntibodies, Compositions, Methods and Uses,” the contents of which arehereby incorporated herein by this reference. In another embodiment, thetherapeutic agent is a protein or enzyme, such as BSA or lysozyme.

Alternatively, the therapeutic agent may be selected from the groupconsisting of baclofen, glial-cell line-derived neurotrophic factor, aneurotrophic factor, conatonkin G, Ziconotide, clonidine, axokine, anantisense oligonucleotide, adrenocorticotropic hormone, angiotensin I,angiotensin II, atrial natriuretic peptide, B-natriuretic peptide,bombesin, bradykinin, calcitonin, cerebellin, dynorphin N, alphaendorphin, beta endorphin, endothelin, enkephalin, epidermal growthfactor, fertirelin, follicular gonadotropin releasing peptide, galanin,glucagon, glucagon-like peptido-1, gonadorelin, gonadotropin, goserelin,growth hormone releasing peptide, histrelin, human growth hormone,insulin, an alpha-, beta-, or omega-interferon, Nesiritide, leuprolide,luteinizing hormone-releasing hormone, motilin, nafarelin, neurotensin,oxytocin, relaxin, somatostatin, substance P, tumor necrosis factor,triptorelin, vasopressin, growth hormone, nerve growth factor, a bloodclotting factor, and a ribozyme. In one embodiment, the at least onesolvent is benzyl benzoate, the at least one polymer ispolyvinylpyrrolidone, and the active agent is omega-interferon(omega-INF). The active agent may also be selected from small moleculessuch as, for example, ocaperidone, risperidone, and paliperidone.

The therapeutic agent may be formulated into particles having a particlesize that ranges from approximately 0.1 μm to approximately 250 μm. Theparticles of the therapeutic agent may be produced by conventionalprocesses including, but not limited to, mechanical milling and sievingor spray drying. To provide additional stability to the therapeuticagent, a stabilizer may, optionally, be present in the injectable,nonaqueous suspension. The stabilizer of the therapeutic agent may be asugar, such as sucrose, trehalose, sorbitol, mannitol, a monosaccharidealcohol, or mixtures thereof. If the therapeutic agent is a protein, thetherapeutic agent and, optionally, the stabilizer may be dissolved intoa solution, which is lyophilized to produce particles of the therapeuticagent. These particles are ground and sieved to the desired particlesize. Alternatively, the solution may be spray-dried orspray-freeze-dried to produce particles of the desired size. In additionto the stabilizer, the formulation of the therapeutic agent may,optionally, include at least one pH modifier.

The single component vehicle may be a single amphiphilic material havinga liquid to semi-solid form. The amphiphilic material may be a solvent,surfactant, or excipient. As used herein, the term “single componentvehicle” refers to a one-component vehicle. As such, the singlecomponent vehicle does not include additional solvents, surfactants, orexcipients, which simplifies formulation of the injectable, nonaqueoussuspension. As used herein, the term “amphiphilic” refers to a compoundhaving a polar, water-soluble group attached to a nonpolar,water-insoluble group or chain. The term “liquid to semi-solid” refersto a material having intermediate properties, such as viscosity, betweenthose of a solid and a liquid. The viscosity of the single componentvehicle may be selected to provide the injectable, nonaqueous suspensionwith a low injection force when administered to a human or animal. Thesingle component vehicle may have a melting point or a pour point ofapproximately room temperature (approximately 25° C.), so that thesingle component vehicle is a liquid or semi-solid at body temperature.

Examples of amphiphilic materials that may be used as the singlecomponent vehicle include, but are not limited to, a polyethoxylatedcastor oil or derivative thereof (collectively referred to herein as a“polyethoxylated castor oil”), a polyoxyethylene alkyl ether, apolyoxyethylene sorbitan fatty acid ester, a polyoxyethylene stearate, ablock copolymer of polyethylene oxide (“PEO”)-polypropylene oxide(“PPO”)-PEO, a block copolymer of PPO-PEO-PPO, a tetra-functional blockcopolymer of PEO-PPO, such as (PEO-PPO)₂-(PPO-PEO)₂, or atetra-functional block copolymer of PPO-PEO, such as(PPO-PEO)₂-(PEO-PPO)₂. In one embodiment, the single component vehicleis a polyethoxylated castor oil.

One example of a polyethoxylated castor oil is sold under the tradenameCremophor® and is commercially available from BASF Corp. (Mount Olive,N.J.). Cremophor® products of various purities and viscosities areproduced by BASF Corp. and may be used in the present invention, such asCremophor® A 25, Cremophor® A 6, Cremophor® EL, Cremophor® RH 40,Cremophor® ELP, or mixtures thereof. Cremophor® ELP and Cremophor® ELare nonionic solubilizers and emulsifiers produced by reacting castoroil with ethylene oxide in a molar ratio of 1 to 35. Cremophor® RH 40 isa nonionic solubilizer and emulsifier produced by reacting castor oilwith ethylene oxide in a molar ratio of 1 to 45. While the compositionof Cremophor® products is proprietary, it is believed that the reactionproduct of castor oil and ethylene oxide includes a complex mixture ofunmodified castor oil and a variety of polyethylene glycols,polyethoxylated glycerol, polyethoxylated fatty acids, and mono-, di-and triesters of glycerol that are polyethoxylated to differing degrees.In one embodiment, the single component vehicle is Cremophor® ELP, whichhas a viscosity of between approximately 600 mPa·sec and approximately750 mPa·sec at 25° C. In another embodiment, the single componentvehicle is Cremophor® EL, which has a viscosity of between approximately700 mPa·sec and approximately 800 mPa·sec at 25° C. In anotherembodiment, the single component vehicle is Cremophor® RH 40. A 30% aaqueous solution of Cremophor® RH 40 has a Hoeppler viscosity at 25° C.of between approximately 20 mPa·sec and approximately 40 mPa·sec.

Examples of polyoxyethylene alkyl ethers that may be used as the singlecomponent vehicle include, but are not limited to, Brij® 35, Brij® 52,Brij® 56, Brij® 93, Brij® 97, Brij® 99, Ethylan® 256, Ethylan® 257,Ethylan® 2512, Renex® 30, Renex® 31, Texofor AP, Texofor A6, TexoforA10, or mixtures thereof. Examples of polyoxyethylene sorbitan fattyacid esters include, but are not limited to, polysorbate 61, polysorbate65, polysorbate 80, or mixtures thereof. Examples of polyoxyethylenestearates include, but are not limited to, polyoxyl 6 stearate, polyoxyl8 stearate, polyoxyl 12 stearate, polyoxyl 20 stearate, polyoxyl 40stearate, polyoxyl 12 distearate, or mixtures thereof. Examples ofPEO-PPO-PEO include, but are not limited to, Pluronic® L44, Pluronic®L64, Pluronic® L122, Pluronic® P65, Pluronic® P75, Pluronic® P84,Pluronic® P85, Pluronic® P103, Pluronic® P104, Pluronic® P105, Pluronic®P123, or mixtures thereof. Examples of PPO-PEO-PPO include, but are notlimited to, Pluronic® R 10R5, Pluronic® 17R4, Pluronic® 22R4, Pluronic®25R4, Pluronic® 25R5, Pluronic® 31R4, or mixtures thereof. Examples of(PEO-PPO)-(PPO-PEO)_(z) include, but are not limited to, Tetronic® 704,Tetronic®904, Tetronic® 1104, or mixtures thereof. Examples of(PPO-PEO)₂-(PEO-PPO)₂ include, but are not limited to, Tetronic® R 50R8,Tetronic® 90R4, Tetronic® 150R4, or mixtures thereof.

A high concentration of the therapeutic agent may be suspended in thesingle component vehicle, enabling delivery of the therapeutic agent ina relatively small volume. For instance, the therapeutic agent may bepresent in the injectable, nonaqueous suspension at a concentration ofup to approximately 500 mg/ml. For instance, the therapeutic agent maybe present in the injectable, nonaqueous suspension at a concentrationthat ranges from greater than or equal to approximately 50 mg/ml toapproximately 500 mg/ml. Such high concentrations of therapeutic agentwould not be achievable in an aqueous formulation. Since the therapeuticagent is present at such a high concentration, the injectable,nonaqueous suspension may be used to deliver a therapeutic agent thathas a low potency.

In a particular embodiment, the therapeutic agent may account for fromapproximately 5% by weight (“wt %”) of a total weight of the injectable,nonaqueous suspension to approximately 50 wt % of the total weight ofthe injectable, nonaqueous suspension. For instance, the therapeuticagent loading may range from approximately 10 wt % of the total weightof the injectable, nonaqueous suspension to approximately 50 wt % of thetotal weight of the injectable, nonaqueous suspension.

The injectable, nonaqueous suspension may be produced by mixing theparticles of the therapeutic agent into the single component vehicle bytechniques known in the art. Since the therapeutic agent and the singlecomponent vehicle are combined using conventional techniques, productionof the injectable, nonaqueous suspension is not described in detailherein. The particles of the therapeutic agent may be substantiallyhomogeneously dispersed in the single component vehicle, producing asubstantially homogeneous injectable, nonaqueous suspension.

The injectable, nonaqueous suspension may, optionally, include a smallamount of at least one antioxidant. The antioxidant may be d-alphatocopherol acetate, dl-alpha tocopherol, ascorbyl palmitate, butylatedhydroxyanidole, ascorbic acid, butylated hydroxyanisole,butylatedhydroxyquinone, butylhydroxyanisol, hydroxycomarin, butylatedhydroxytoluene, cephalm, ethyl gallate, propyl gallate, octyl gallate,lauryl gallate, propylhydroxybenzoate, trihydroxybutylrophenone,dimethylphenol, diterlbulylphenol, vitamin E, lecithin, ethanolamine, ormixtures thereof. If an antioxidant is present in the injectable,nonaqueous suspension, the antioxidant may be premixed with thetherapeutic agent before mixing with the single component vehicle.Alternatively, the antioxidant may be premixed with the single componentvehicle before mixing with the therapeutic agent or may be loadedseparately into the single component vehicle.

The injectable, nonaqueous suspension may be preloaded in a syringe and,therefore, is injection ready without mixing or reconstitution. Theinjectable, nonaqueous suspension may be loaded into the syringe byconventional techniques, which are well known in the art and, thus, arenot described in detail herein. Since the therapeutic agent remains in asubstantially solid form, the injectable, nonaqueous suspension can havea long shelf life stability. The injectable, nonaqueous suspension maybe fluidly injectable at 25° C. The injectable, nonaqueous suspension inthe preloaded syringe may be injected by hand. Alternatively, thepreloaded syringe may be used with an autoinjector, where injection ofthe injectable, nonaqueous suspension is powered by the mechanical forceof the autoinjector.

A dosage kit that includes the injectable, nonaqueous suspension and atleast one syringe is also disclosed. In one embodiment, the syringe isan auto-injector syringe. In another embodiment, the syringe is dividedsuch that the therapeutic agent and the single component vehicle remainseparate until being mixed before injection. In another embodiment, twosyringes are provided in the dosage kit. One syringe may contain thetherapeutic agent while the other syringe may contain the singlecomponent vehicle. The contents of the first and second syringes may becombined before injection. The injectable, nonaqueous suspension may beadministered to a patient or animal by any route, such as by SC or IMadministration. Since the single component vehicle has a low viscosity,the injectable, nonaqueous suspension may have a low injection force.For instance, the injection force may be less than or equal toapproximately 50 lbf; such as less than or equal to approximately 30 lbfor less than or equal to approximately 20 lbf.

The following examples serve to explain embodiments of the presentinvention in more detail. These examples are not to be construed asbeing exhaustive or exclusive as to the scope of this invention.

EXAMPLES

In vitro release experiments were conducted to test suspensionformulations to demonstrate release of the therapeutic agent into abuffer solution. The in vitro release characteristics are related to invivo release characteristics. Injection force experiments were performedto test the injectability of the suspension formulations becauseinjectability is an important performance characteristic. Stability isalso an important performance characteristic for suspensionformulations. Therefore, sampling was conducted at extended timeintervals to demonstrate stability of the therapeutic agent.Biocompatibility tests were also conducted to demonstrate tolerance tothe suspension injection in a rat model.

Example 1 Preparation of Lysozyme Particles

A lysozyme solution was prepared by dissolving lysozyme (available fromSigma-Aldrich Corp. (St. Louis, Mo.)) in 6.5 mM sodium phosphate buffer,pH 6.0, at a protein concentration of 65 mg/mL. Sucrose and Tween 80 (orpolysorbate 80) were optionally added to the lysozyme solution with theconcentration of sucrose and Tween 80 in the final solution of 0%-5.5%and 0%-0.0065% weight/volume, respectively. The lysozyme solution waslyophilized according to the conditions shown in Table 1.

TABLE 1 Lyophilization Conditions for the Preparation of LysozymeParticles. Chamber Pressure Hold Time Process Step Shelf Temperature (°C.) (mBar) (hours) Loading  +5 N/A 2 Freezing −50 (rate 0.5° C.) N/A 2Freezing −50 N/A 2.5 Vacuum on −50 120 mT  0.5 Vacuum hold −50 120 mT 0.5 1° Drying −10 (rate 1° C./minute) 120 mT  0.75 1° Drying −10 120 mT 24 2° Drying  0 (rate 0.1° C./minute) 80 mT 1.7 2° Drying  0 80 mT 2 2°Drying +35 (rate 0.25° C./minute) 80 mT 2.3 2° Drying +35 80 mT 10 2°Drying +20 (rate 1° C./minute) 80 mT 0.25 2° Drying +20 80 mT 2 MinimumTotal time = 50.5 hours

Lysozyme particles having the desired particle size were prepared bygrinding the lyophilized lysozyme in a Waring blender and passing theparticles through a series of sieves having determined mesh sizes.Lysozyme particles having a particle size of less than approximately 38μm, between from approximately 38 μm to approximately 63 μm, less thanapproximately 125 μm, and less than approximately 250 μm were producedby this process. Alternatively, lysozyme particles were prepared bydiluting the lyophilized lysozyme described above to a concentration ofapproximately 20 mg/ml with deionized water and spray drying thesolution, resulting in lysozyme particles having a particle size thatranged from approximately 1 μm to approximately 10 μm.

Example 2 Preparation of BSA Particles

BSA particles were produced according to the methods described inExample 1, except hat BSA was used in place of lysozyme.

Example 3 Preparation of CNTO 1275 Particles

Particles of a mAb were produced by dissolving CNTO 1275 in 6.5 mMsodium phosphate buffer with sucrose (concentration of 5.5% w/v), pH6.0, with a protein concentration of 65 mg/mL. To the solution of CNTO1275 was added 0.0065% w/v of Tween 80 (or polysorbate 80). The solutionwas lyophilized according to the conditions shown in Table 2.

TABLE 2 Lyophilization Conditions for Preparation of CNTO 1275Particles. Chamber Pressure Hold Time Process Step Shelf Temperature (°C.) (mBar) (hours) Loading  +5 N/A 2 Freezing −50 (rate 0.5° C.) N/A 2Freezing −50 N/A 2.5 Vacuum on −50 120 mT  0.5 Vacuum hold −50 120 mT 0.5 1° Drying −10 (rate 1° C./minute) 120 mT  0.75 1° Drying −10 120 mT 24 2° Drying  0 (rate 0.1° C./minute) 80 mT 1.7 2° Drying  0 80 mT 2 2°Drying +35 (rate 0.25° C./minute) 80 mT 2.3 2° Drying +35 80 mT 10 2°Drying +20 (rate 1° C./minute) 80 mT 0.25 2° Drying +20 80 mT 2 MinimumTotal time = 50.5 hours

CNTO 1275 particles having a particle size of less than approximately125 μm were prepared by grinding the lyophilized formulation in a Waringblender and sieving through a 120 mesh sieve.

Example 4 Preparation of Nonaqueous Suspensions

Nonaqueous suspensions of lysozyme, BSA, or CNTO 1275 particles wereprepared. The particles (lysozyme, BSA, or CNTO 1275), which wereprepared as described in Examples 1-3, were mixed with Cremophor® ELP orCremophor® RH 40 using an overhead mixer. The mixing was performed atroom temperature inside a dry box. The particles and Cremophor® ELP orCremophor® RH 40 were weighed and transferred into a 25 cc glasssyringe. The particle loading was from approximately 20 wt % toapproximately 50 wt %, resulting in a protein concentration in a finalformulation of from approximately 120 mg/ml to approximately 500 mg/mL.An electric stirrer with a stainless steel spatula blade was used toblend the particles into the Cremophor® ELP or Cremophor® RH 40 at fromapproximately 50 rpm to approximately 300 rpm for approximately 5minutes. The suspensions were filled into glass injection syringes,yielding an injection ready dosage form. The suspensions were stored ata refrigerated temperature (approximately 4° C. to approximately 5° C.)prior to injection.

Example 5 In Vitro Release Testing of a BSA/Cremophor® ELP Formulation

Spray-dried BSA particles were mixed with Cremophor® ELP at a ratio of37 wt % BSA particles and 63 wt % Cremophor® ELP. Three 50-60 mg samplesof the BSA/Cremophor® ELP suspension were placed in vials along with PBSbuffer solution. The vials were placed on a shaker in a 37° C. oven.Samples of the supernatant were removed from the vials at various timepoints to determine the BSA content, and fresh PBS solution of the same(removed) volume was added back to the vials. The supernatant wasanalyzed using UV Spectroscopy to determine the BSA content. A graph ofthe cumulative protein released versus time is shown in FIG. 1.Approximately 95% of the BSA was released from the suspension withinapproximately 20 minutes of the test initiation, indicating the proteinin the suspension of Cremophor® ELP was released into the mediuminstantaneously. As such, the Cremophor® ELP does not trap thetherapeutic agent to cause a depot effect.

Example 6 Injection Force Testing of BSA/Cremophor® ELP Formulation

Injectability of a BSA/Cremophor® ELP suspension was determined bymeasuring the force required to push the suspension through a fine gaugeneedle. Cremophor® ELP and BSA particles having a particle size of lessthan or equal to approximately 125 μm were blended in a ratio of 60:40by weight, respectively. The suspension was loaded into three Hamilton500 μl Gastight® syringes. The syringes were filled with approximately0.4 cc of the suspension and aluminum hub hypodermic needles (21-gauge,1 inch (2.54 cm) in size) were placed on each syringe. The syringes werepackaged, three syringes to each polyethane bag, and stored in therefrigerator at approximately 5° C. At 1-, 5-, 28-, and 36-week timeperiods, samples were removed from the refrigerator for injection forcetesting. The syringes were equilibrated to room temperature(approximately 25° C.) for at least two hours before testing. Theinjection rate was set at approximately 1 ml/minute, which is equivalentto a crosshead speed of 4.7 inch (11.938 cm)/minute. The injectiontesting was conducted at room temperature and the peak (maximum) forcevalue was recorded. Injection force testing was carried out on a Mini-55Instron tensile testing instrument.

The injection force at room temperature versus storage time at 5° C. forthe BSA/Cremophor® ELP suspension is shown in FIG. 2. No increase in theforce required to dispense the BSA/Cremophor® ELP suspension wasobserved as a function of storage time, which demonstrates that theBSA/Cremophor® ELP suspension is physically stable in the described timeframes. No settling of the BSA particles in the Cremophor® ELP wasobserved.

Example 7 Injection Force Testing of BSA/Cremophor® RH 40 Suspension

Injectability of a BSA/Cremophor® RH 40 suspension was determined bymeasuring the force required to push the suspension through a fine gaugeneedle. Cremophor® RH 40 and BSA particles having a particle size ofless than or equal to approximately 125 μm were blended in a ratio of60:40 by weight, respectively. The suspension was loaded into threeHamilton 500 μl Gastight® syringes. The syringes were filled withapproximately 0.4 cc of the suspension and aluminum hub hypodermicneedles (21-gauge, 1 inch (2.54 cm) in size) were placed on eachsyringe. The syringes were packaged, three syringes to each polyethanebag, and stored in the refrigerator at approximately 5° C. Samples wereremoved from the refrigerator at 5 days and at 180 days for theinjection force testing. The syringes were equilibrated to roomtemperature for at least two hours before the testing. The injectionrate was set at approximately 1 ml/minute, which is equivalent to acrosshead speed of 4.7 inch (11.938 cm)/minute. The injection testingwas conducted at room temperature and the peak (maximum) force value wasrecorded. Injection force testing was carried out on a Mini-55 Instrontensile testing instrument.

The injection force at room temperature versus storage time at 5° C. forthe BSA/Cremophor® RH 40 suspension is shown in FIG. 3. No increase inthe force required to dispense the BSA/Cremophor® RH 40 suspension wasobserved as a function of storage time, which demonstrates that theBSA/Cremophor® RH 40 suspension is physically stable in these timeframes. No settling of the BSA particles in the Cremophor® RH 40 wasobserved.

Example 8 Stability of CNTO 1275/Cremophor® ELP Formulation

Cremophor® ELP was cleaned with aluminum oxide powder to reduce theperoxide level and was passed through sterile, 0.2 μm PTFE filters. Two10 mg portions of CNTO 1275 were weighed into vials for each stabilitytime point. Into each vial, 0.1 ml of cleaned Cremophor® ELP was added.The CNTO 1275/Cremophor® ELP samples were then mechanically stirred andcaps were placed on each vial. The vials were removed from the dry boxand placed in a refrigerator at 5° C. CNTO 1275 particles, withoutCremophor® ELP, were stored with the suspension samples as a control. Acontrol formulation that included 25% polyvinylpyrollidone (“PVP”) and75% ultrapure polyethylene glycol 400 (“PEG 400”) was also stored withthe suspension samples. At 0-, 1-, 4-, 8-, and 12-week intervals,samples were removed from the refrigerator and tested for monomercontent by size exclusion chromatograph (“SEC.”)

A graph of monomer content versus storage time at 5° C. is shown in FIG.4. The results show that CNTO 1275 in Cremophor® ELP (labeled“Cremophor® ELP” in FIG. 4) had the same monomer content as CNTO 1275particles without Cremophor® ELP (labeled “CNTO 1275 Particles” in FIG.4) at each time point, which demonstrates that the Cremophor® ELPvehicle did not degrade the CNTO 1275 monoclonal antibody within 12weeks of storage time at 5° C. In comparison, significant proteinaggregation was observed with the formulation that included the CNTO1275 particles, 25% PVP, and 75% PEG 400, which is shown by the reducedmonomer content.

Example 9 Biocompatibility of Cremophor® ELP

Cremophor® ELP was cleaned with aluminum oxide powder to reduce theperoxide level and was passed through sterile, 0.2 μm PTFE filters. Thecleaned Cremophor® ELP was transferred into 0.25 ml autoclaved glasssyringes. Each syringe was filled with between 0.155 ml and 0.160 ml ofCremophor® ELP. The syringes were fitted with 23 gauge, 1-inch (2.54 cm)aluminum hub hypodermic needles to deliver a 0.100 ml subcutaneousinjection into rats. The Cremophor® ELP was administered at sixinjection sites. The injection sites were qualitatively assessed forswelling and irritation each day for two weeks. Injection sites werescored as “0” for no swelling and irritation, “1” for minimal swellingand irritation, “2” for mild swelling and irritation, “3” for moderateswelling and irritation, and “4” for severe swelling and irritation.Suspension vehicles of PVP/benzyl benzoate (“BB”) (30%/70%) and laurylalcohol (“LA”) were used as controls. A graph of the average severityscore versus study day is shown in FIG. 5. The Cremophor® ELP injectionsshowed no swelling at the rat injection sites. The Cremophor® ELPreceived the best test score of “0” each of the 14 days that theinjection sites were observed. In comparison, minimal to moderateswelling at the injection sites were observed with the PVP/BB and LAcontrols.

While the invention may be susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and have been described in detail herein.However, it should be understood that the invention is not intended tobe limited to the particular forms disclosed. Rather, the invention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the followingappended claims.

What is claimed is:
 1. An injectable, nonaqueous suspension comprising at least one therapeutic agent suspended in a single component vehicle having an amphiphilic material.
 2. The injectable, nonaqueous suspension of claim 1, wherein the single component vehicle comprises a polyethoxylated castor oil or derivative thereof, a polyoxyethylene alkyl ether, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene stearate, a block copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, a block copolymer of polypropylene oxide-polyethylene oxide-polypropylene oxide, a tetra-functional block copolymer of polyethylene oxide-polypropylene oxide, or a tetra-functional block copolymer of polypropylene oxide-polyethylene oxide.
 3. The injectable, nonaqueous suspension of claim 1, wherein the single component vehicle comprises a reaction product of castor oil with ethylene oxide in a molar ratio of 1 to 35 or a reaction product of castor oil with ethylene oxide in a molar ratio of 1 to
 45. 4. The injectable, nonaqueous suspension of claim 1, wherein the at least one therapeutic agent comprises a small molecule, protein, antibody, mimetibody, monoclonal antibody, antibody fragment, peptide, nucleotide, DNA fragment, RNA fragment, plasmid fragment, nucleotide fragment, or mixtures thereof.
 5. The injectable, nonaqueous suspension of claim 1, wherein the at least one therapeutic agent is present at a concentration that ranges from greater than or equal to approximately 50 mg/ml to approximately 500 mg/ml.
 6. The injectable, nonaqueous suspension of claim 1, wherein the at least one therapeutic agent comprises from approximately 5% by weight of a total weight of the injectable, nonaqueous suspension to approximately 50% by weight of the total weight of the injectable, nonaqueous suspension.
 7. The injectable, nonaqueous suspension of claim 1, further comprising at least one antioxidant.
 8. The injectable, nonaqueous suspension of claim 1, wherein the suspension has an injection force that is less than or equal to about 50 lbf.
 9. The injectable, nonaqueous suspension of claim 1, wherein the single component vehicle comprises a polyethoxylated castor oil or derivative thereof and the at least one therapeutic agent comprises a monoclonal antibody.
 10. A dosage kit comprising a syringe and an injectable, nonaqueous suspension, the injectable, nonaqueous suspension comprising at least one therapeutic agent suspended in a single component vehicle having an amphiphilic material.
 11. The dosage kit of claim 10, wherein the single component vehicle comprises a polyethoxylated castor oil or derivative thereof, a polyoxyethylene alkyl ether, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene stearate, a block copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, a block copolymer of polypropylene oxide-polyethylene oxide-polypropylene oxide, a tetra-functional block copolymer of polyethylene oxide-polypropylene oxide, or a tetra-functional block copolymer of polypropylene oxide-polyethylene oxide.
 12. The dosage kit of claim 10, wherein the at least one therapeutic agent comprises a small molecule, protein, antibody, mimetibody, monoclonal antibody, antibody fragment, peptide, nucleotide, DNA fragment, RNA fragment, plasmid fragment, nucleotide fragment, or mixtures thereof.
 13. A method of administering a therapeutic agent, comprising: suspending at least one therapeutic agent in a single component amphiphilic vehicle to form an injectable, nonaqueous suspension; and injecting the injectable, nonaqueous suspension into a patient in need thereof.
 14. The method of claim 13, wherein suspending at least one therapeutic agent in a single component vehicle comprises suspending the at least one therapeutic agent in a single component vehicle selected from the group consisting of a polyethoxylated castor oil or derivative thereof, a polyoxyethylene alkyl ether, a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene stearate, a block copolymer of polyethylene oxide-polypropylene oxide-polyethylene oxide, a block copolymer of polypropylene oxide-polyethylene oxide-polypropylene oxide, a tetra-functional block copolymer of polyethylene oxide-polypropylene oxide, and a tetra-functional block copolymer of polypropylene oxide-polyethylene oxide.
 15. The method of claim 13, wherein suspending at least one therapeutic agent in a single component vehicle comprises suspending the at least one therapeutic agent in a reaction product of castor oil with ethylene oxide in a molar ratio of 1 to 35 or in a reaction product of castor oil with ethylene oxide in a molar ratio of 1 to
 45. 16. The method of claim 13, wherein suspending at least one therapeutic agent in a single component vehicle comprises suspending a small molecule, protein, antibody, mimetibody, monoclonal antibody, antibody fragment, peptide, nucleotide, DNA fragment, RNA fragment, plasmid fragment, nucleotide fragment, or mixtures thereof in the single component vehicle.
 17. The method of claim 13, wherein the at least one therapeutic agent is present at a concentration that ranges from greater than or equal to approximately 50 mg/ml to approximately 500 mg/ml.
 18. The method of claim 13, wherein the at least one therapeutic agent comprises from approximately 5% by weight of a total weight of the injectable, nonaqueous suspension to approximately 50% by weight of the total weight of the injectable, nonaqueous suspension.
 19. The method of claim 13, further comprising adding at least one antioxidant to the injectable, nonaqueous suspension.
 20. The method of claim 13, wherein the single component amphiphilic vehicle comprises a polyethoxylated castor oil or derivative thereof and the at least one therapeutic agent comprises a monoclonal antibody. 